Endothelial Rbpj deletion normalizes Notch4-induced brain arteriovenous malformation in mice

Nielsen and Zhang et al. show that well-established, Notch4-induced brain arteriovenous malformations are normalized, following deletion of the Notch signaling mediator, Rbpj. Upon complete regression, virtually no AVM relapses in adults, even when the causal factor is reintroduced.

Inducible expression of constitutively active Notch4, in postnatal endothelium, termed Notch4* tetEC transgene herein, leads to AVM and moribundity by postnatal day (P) 36 in mice (Murphy et al., 2008). Notch4* tetEC is controlled by the tetracycline (tet)-repressible system, such that administration of tet represses Notch4* tetEC expression. Turning off the causal Notch4* tetEC transgene elicits bAVM regression (Murphy et al., 2012) and extends survival (Murphy et al., 2008) in mice, normalizing the established AVM to microvessels. Here, we tested a new treatment strategy that inhibits downstream Notch signaling without switching off the causal Notch4* tetEC transgene. We deleted Rbpj, a mediator of Notch, and showed regression of established bAVMs in Notch4* tetEC mice.

Results and discussion
Expression of Notch4* tetEC from birth led to well-established AVM with minimal animal illness by P16 As AVM pathogenesis was well established in 100% of P16 Tie-tTA; TRE-Notch4* (Notch4* tetEC ) brains, in which tet was withdrawn from birth and thus Notch4* tetEC was induced from birth, we chose P16 as the timepoint to induce AVM regression. Another advantage of P16 is that fewer mice had reached moribundity by this time, thus offering an opportunity to achieve AVM rescue in "healthier" mice, with fewer confounding effects of general illness. Furthermore, we considered the time it takes to achieve effective inhibition of Notch4* signaling and to induce AVM regression, following tamoxifen (TAM) injection at P16 and subsequent Rbpj deletion. After TAM administration, it takes time for Cre to be expressed and to excise the floxed Rbpj sequence, time for the cells to stop producing Rbpj, and time for the already-produced Rbpj to be degraded and cleared from cells, before Rbpj deletion is effectively achieved. Similar to how Rbpj is cleared from the cells, it also takes time to stop transmission of Notch4* tetEC signaling and for the downstream targets to be cleared, and to achieve effective inhibition of Notch4* tetEC signaling. Thus, the actual timing to abrogate Notch4* tetEC signaling is after P16, when the Notch4* tetEC mice had progressed into more mature AVM, and when more Notch4* tetEC mice began to reach moribundity from ∼P18 (Murphy et al., 2008). Thus, TAM administration from P16 is an optimal time point to induce the regression of well-established AVMs.
We thus documented AVM pathogenesis at P16 as a reference point for AVM regression (Fig. S1, A and B). In all Notch4* tetEC mice examined, mean brain AV diameter and the proportion of AV connections with diameter ≥12.5 μm were increased in Notch4* tetEC mice (Fig. 1, A-C, and Fig. S1 C). To test for functional AV shunting, we performed a microsphere passage assay and found FITCmicrospheres confined to brain capillaries in controls but lodged in lungs in Notch4* tetEC mutants, indicating microsphere circulation through AV shunts by P16 (Fig. 1, D and E9).
Other features of bAVM developed by P16 in mice with Notch4* tetEC expressed from birth. Morphologically, casts of brain vessels appeared more tortuous in Notch4* tetEC vs. controls (Fig. 1, G-I), with AVM niduses emerging in Notch4* tetEC brains at P16. Perfused Notch4* tetEC brains, but not controls, showed evidence for hemorrhage by P16 (Fig. 1, J-L). Histological analysis revealed minor tissue lesions in Notch4* tetEC brains by P16 (Fig. 1, M-O). Finally, we detected hypoxic cells in brain parenchyma in Notch4* tetEC mice by P16, but not in controls ( Fig. 1, P-R). We consistently observed bleeding and tortuous vessels in the cerebellum. Similarly, mice with mutations in genes responsible for cerebral cavernous malformations also develop cerebral cavernous malformations in mouse cerebellum (Zhou et al., 2016). The cerebellum is a region that undergoes extensive morphogenesis in immature brains (Acker et al., 2001;Chapman et al., 2022), suggesting that cerebellar endothelial cells (ECs) may be regionally and temporally susceptible to Notch4* tetEC activation.
To accommodate increased blood flow through AV shunts and related systemic hemodynamic changes, the heart must compensate by increasing cardiac output and may develop compensatory cardiomegaly or ventricular hypertrophy (Carlson et al., 2005). Despite developing hallmarks of AVM by P16, Notch4* tetEC mice did not display cardiomegaly or increased heart weight/body weight percentage (Fig. S1 D). Thus, while we observed several features of bAVM by P16, these likely did not alter systemic hemodynamics enough to lead to compensatory cardiomegaly. Together, our data demonstrate P16 as a time point at which all Notch4* tetEC mice established bAVM pathologies.
Expression of Notch4* tetEC from birth led to severe AVM and compromised health by P21 We next established an experimental time point at P21 when Notch4* tetEC AVM pathologies were more severe. As ∼75% Notch4* tetEC mice reach moribundity by P21 (Murphy et al., 2008), and because surviving Notch4* tetEC mice reach moribundity shortly after P21, we chose this timepoint to study the regression from severe AVMs. Gross morphological analysis and vascular casting revealed evidence of hemorrhage and vessel tortuosity (particularly in the cerebellum) in P21 Notch4* tetEC brain but not controls (Fig. S1, E, F, H, and I). Immunostaining against CD31 and α-smooth muscle actin (αSMA) showed greatly enlarged and tortuous AV connections, with increased expression of the arterial marker αSMA in Notch4* tetEC brains, as compared with controls (Fig. S1, G and J). AV connection diameters were significantly increased in P21 Notch4* tetEC brain as compared with controls ( Fig. S1 K). The mean body weight of Notch4* tetEC mice was less than the negative controls ( Fig. S1 L), and the percentage of heart weight/body weight of Notch4* tetEC mice was greater than the negative controls ( Fig. S1 M). These data suggest that expression of Notch4* tetEC from birth led to severe brain AVM, with compromised health and systemic hemodynamic effects, by P21.
To document overall health, we monitored mice daily for signs of distress, neurological impairment, and ill health, and we harvested brain tissue at moribundity; thus, harvest time points differ among genetic cohorts. We analyzed total body weight at P16 and at moribund. At P16, no differences in body weight were seen (Fig. 2 C). At moribund, Notch4* tetEC had little net change in body weight, similar to Notch4* tetEC ;Rbpj iΔEC-het mice ( Fig. 2 D). By contrast, Notch4* tetEC ;Rbpj iΔEC mice gained significantly more weight than Notch4* tetEC ; however, Notch4*tetEC;Rbpj iΔEC mice did not gain as much weight as negative controls (Rbpj iΔEC-het ; Fig. 2 D). Notably, Rbpj iΔEC mice gained less body weight than negative controls (Fig. 2 D). However, when we tracked body weight changes daily from P16 TAM administration (rather than assessing change between P16 and the moribundity timepoint), we noticed that by P25, Notch4* tetEC ;Rbpj iΔEC mice were gaining weight comparably to Rbpj iΔEC mice ( Fig. S2 B). As a possible cause for decreased body weight gain, we found severe vascular abnormalities associated with the gastrointestinal (GI) tract in all Rbpj iΔEC mice examined. In addition to enlarged, tortuous vessels in all mice analyzed, 37.8% of Rbpj iΔEC mice developed terminal GI ailments-bloody feces, bleeding rectum, prolapsed rectum-that necessitated immediate euthanasia ( Fig. S2 A). These data show that endothelial deletion of Rbpj improved the overall health of Notch4* tetEC mice, while endothelial deletion of Rbpj alone affected animal health at a later stage.
Endothelial deletion of Rbpj from P16 reduced intracerebral hemorrhage and histopathological abnormalities in Notch4* tetEC mice To assess other bAVM features, we performed gross morphological analysis of perfused brains, showing hemorrhages in moribund Notch4* tetEC and Notch4* tetEC ;Rbpj iΔEC-het mice (Fig. 2, E and F), but not Notch4* tetEC ;Rbpj iΔEC , Rbpj iΔEC and negative control brains (Fig. 2, G-I). Histological analysis also revealed red blood cell infiltration of brain parenchyma, or hemorrhage, in Notch4* tetEC and Notch4* tetEC ;Rbpj iΔEC-het mice (Fig. 2, J and K), but not in Notch4* tetEC ;Rbpj iΔEC , Rbpj iΔEC , or negative controls (Fig. 2, L-N). We examined brain parenchyma for evidence of hypoxia, typical of AVM-adjacent tissue, in Notch4* tetEC mice and found that large swaths of hypoxic cells were seen in Notch4* tetEC and Notch4* tetEC ;Rbpj iΔEC-het brain tissue (Fig. 2, O, P9, and U), but not in Notch4* tetEC ;Rbpj iΔEC , Rbpj iΔEC , or negative control tissue (Fig. 2, Q-S9, and U). As non-patent vessels could contribute to hypoxia, we measured the percentage of lectin-perfused brain tissue in all cohorts and found no significant differences (Fig. 2, O-S9, and U). Together, our results show that endothelial deletion of Rbpj from P16 reduced intracerebral hemorrhage and hypoxia in Notch4* tetEC brains. These findings are consistent with normalization of blood flow and restoration of tissue oxygenation, following normalization of AV connections.
As AV shunting can lead to compensatory cardiomegaly, we documented increased heart/body weight ratio in Notch4* tetEC mice when compared with negative controls (Fig. S2 D). Increased heart/body weight ratio was also measured in Notch4* tetEC ;Rbpj iΔEC-het and Notch4* tetEC ;Rbpj iΔEC mice when compared with negative controls. However, heart/body weight in Rbpj iΔEC mice did not differ when compared with negative controls. These results show that heterozygous or homozygous EC-Rbpj deletion did not rescue cardiomegaly in Notch4* tetEC mice and that endothelial deletion of Rbpj alone did not lead to cardiomegaly at moribundity.
Endothelial deletion of Rbpj from P16 normalized arterial marker expression in Notch4* tetEC mice Notch signaling is required for arterial EC identity, and Notch4 tet * EC expression induces abnormal arterial marker expression in AV shunts and veins (Murphy et al., 2012;Murphy et al., 2008). To test whether endothelial deletion of Rbpj restores normal arterial identity in Notch4* tetEC mice, we examined expression of αSMA and Connexin40 (Cx40). In Notch4* tetEC ;Rbpj iΔEC-het mice, both αSMA and Cx40 were expressed in arteries and abnormally expressed in AV shunts and veins (Fig. 3, S-S0). Endothelial deletion of Rbpj from P16 abolished the abnormal αSMA and Cx40 expression in Notch4* tetEC AV connections and veins, while αSMA and Cx40 expression in arteries was maintained (Fig. 3, T-T0), resembling normal αSMA and Cx40 expression in negative controls (Fig. 3, U-U0). These data demonstrate that endothelial deletion of Rbpj from P16 normalized arterial marker expression in Notch4* tetEC mice.
To gauge overall health, we tracked body weight and found that while Notch4* tetEC ;Rbpj iΔEC mice weighed less than Rbpj iΔEC counterparts (Fig. S3 A) at P21, by 3 wk after Rbpj deletion (P42), body weight between the two cohorts was similar, and this similarity continued until P126 when Notch4* tetEC ;Rbpj iΔEC mice reached moribundity. However, overall health was not completely restored in Notch4* tetEC ;Rbpj iΔEC mice. We monitored GI health in both cohorts and found 1-15% of mice with rectal bleeding and/or prolapsed rectum between P21 and P120+ (Fig.  S3 B). Our findings represent a novel proof of concept for targeting a downstream mediator of the causal Notch signaling pathway, even during advanced stages of pathogenesis, and for triggering the regression of AVM in mice. These data also show that functionally, Notch4 activates Notch canonical signaling through Rbpj in vascular endothelium. However, while this study provides a novel strategy by which targeting Notch signaling can normalize AVM pathologies, targeting Rbpj itself is not ideal as it affected animal health at a later stage. This study may inspire future development of strategies to inhibit Notch signaling without compromising animal health.
Kaplan-Meier analysis revealed that 100% of Notch4* tetEC mice were moribund by P36 (Fig. 5 B). By contrast, only 23% Notch4* tetEC mice (tet off/ON/off paradigm) died by P36 (of those, all died shortly after P21 and by P24), indicating that these mice were too sick to be rescued, and 73.68% survived to P120. Once mice survived beyond the initial treatment time, 87.5% (14/16) mice live beyond 120 days (Fig. 5 B). Suppression of Notch4* tetEC from P21 (tet off/ON/off paradigm) permitted body weight gain similar to Tie2-tTA mice (Fig. 5 C). We assessed the degree of bAVM regression by P120, following Notch4* tetEC induction at birth and suppression at P21. Whole brains showed no evidence for hemorrhage in Notch4* tetEC mice (tet off/ON/off paradigm) at P120 as compared with controls (Fig. 5, D and E), and αSMA expression was limited to arterial vessel segments in Notch4* tetEC , as in controls (tet off/ON/off paradigm; Fig. 5, F and G). CD31 + AV connections (Fig. 5, F-H) and percent heart weight/body weight (Fig. 5 I) in Notch4* tetEC were slightly increased when compared with Tie2-tTA controls (tet off/ON/off paradigm). This confirms that suppression of Notch4* tetEC from P21 nearly normalizes severe AVMs by P120. Finally, we asked whether AVMs relapse, following reinduction of Notch4* tetEC expression at P120. To our surprise, 87.5% (7/8) mice exhibited further improvement at P147 with no detectable hemorrhage (Fig. 5, J and K), vascular abnormalities (Fig. 5, L-N), histopathologies (Fig. 5, P-S9), or enlarged heart weight/body weight ratio (Fig. 5 O). At P138, one mouse was found non-responsive, and casting did not show obvious AVMs in the cortex, though evidence of abnormal vessels and hemorrhage was found in the cerebellum (Fig. S3 C). It is possible that brain tissues were not completely normalized at P120 in this case. Thus, a longer recovery was needed to achieve continued health in this mouse. These results suggest that AVM was not reinitiated after P120, following recovery from P21. Our data show that removal of the causal transgene resolved pathologies associated with Notch4* tetEC AVM and that after near-complete resolution of pathologies, even if the causal transgene was switched back on, there is no AVM relapse. These data are also consistent with our previous finding that expression of Notch4* tetEC in immature, but not mature, ECs induces hallmarks of bAVM (Carlson et al., 2005) and support the idea that mature brain vasculature is not susceptible to Notch4* tetEC -induced AVM formation.
Collectively, our findings make an important advance, demonstrating both AVM regression and tissue reperfusion in Notch4* tetEC mice by targeting a downstream signaling component or by causal transgene reversal. These data show that endothelial Rbpj is critical for the initiation and maintenance of Notch4* tetEC -induced brain AVM in mice and that endothelial Rbpj may be targeted during different stages of AVM pathogenesis. After complete recovery, reintroducing the causal gene does not lead to relapse later in life.

Vascular casting
Casts of brain vasculature were prepared as follows: anesthetized mice were exsanguinated by transcardial perfusion of PBS; MICROFIL:diluent:curing agent (4:5:1) was transcardially perfused; MICROFIL cast cured at room temperature for 45 min; brain tissue was harvested, imaged, or dehydrated in ethanol series, and cleared in methyl salicylate, according to manufacturer's instructions. Images were captured using dissection scope and Leica LAS software.
Microsphere passage assay Mice were anesthetized using isoflurane/oxygen and the left common carotid artery was surgically exposed. 75 μl of 15 μm FluoSpheres, green (450-480 nm; Invitrogen) were injected directly into the left common carotid artery and circulated for 1 min. Brain and lung tissues were harvested and imaged using a fluorescent/brightfield dissection microscope (Leica) and Mi-croManager software.

Gross morphology and histology
For whole-brain imaging to assess indication of hemorrhage, anesthetized mice were exsanguinated by transcardial perfusion of PBS, followed by 1% PFA. Brains were harvested and imaged using a dissection microscope and Leica LAS software. Standard H&E staining on paraffin-embedded tissue sections was performed in the lab and by the Gladstone Institutes Histology and Light Microscopy Core. Images were captured using an upright light microscope and ZEN software (Zeiss).

Quantification using ImageJ
ImageJ software was used to measure (1) diameters of AV connections at their narrowest; (2) hypoxyprobe+ area per total area of tissue examined; and (3) perfused lectin+ area per total area of tissue section examined. Measurement data were acquired from 12-μm-thick tissue sections.

Statistical analysis
Data are shown as mean ± SD. An unpaired Student's t test with Welch's correction or one-way ANOVA with Tukey's multiple comparison test was used to analyze variance among experimental groups. P < 0.05 was considered significant. Data in the figures are annotated as follows: *P < 0.05; **P < 0.01; and ***P < 0.001. Prism software (Graph Pad) or R Statistical Software was used to generate data graphs and perform statistical analyses.